Method for treating ocular hypertension

ABSTRACT

This invention relates to the use of potent potassium channel blockers or a formulation thereof in the treatment of glaucoma and other conditions related to elevated intraoccular pressure in the eye of a patient. This invention also relates to the use of such compounds to provide a neuroprotective effect to the eye of a mammalian species, particularly humans.

This application claims the benefit of Provisional Application No.60/176,694, filed Jan. 18, 2000.

This is a division of application Ser. No. 09/765,716 filed Jan. 17,2001 now U.S. Pat. No. 6,548,535.

BACKGROUND OF THE INVENTION

Glaucoma is a degenerative disease of the eye wherein the intraocularpressure is too high to permit normal eye function. Damage eventuallyoccurs to the optic nerve head, resulting in irreversible loss of visualfunction. If untreated, glaucoma may eventually lead to blindness.Elevated intraocular pressure or ocular hypertension, is now believed bythe majority of ophthalmologists to represent the earliest phase in theonset of glaucoma.

Many of the drugs formerly used to treat glaucoma proved unsatisfactory.The early methods of treating glaucoma employed pilocarpine and producedundesirable local effects that made this drug, though valuable,unsatisfactory as a first line drug. More recently, clinicians havenoted that many β-adrenergic antagonists are effective in reducingintraocular pressure. While many of these agents are effective for thispurpose, there exist some patients with whom this treatment is noteffective or not sufficiently effective. Many of these agents also haveother characteristics, e.g., membrane stabilizing activity, that becomemore apparent with increased doses and render them unacceptable forchronic ocular use and can also cause cardiovascular effects.

Although pilocarpine and β-adrenergic antagonists reduce intraocularpressure, none of these drugs manifests its action by inhibiting theenzyme carbonic anhydrase, and thus they do not take advantage ofreducing the contribution to aqueous humor formation made by thecarbonic anhydrase pathway.

Agents referred to as carbonic anhydrase inhibitors decrease theformation of aqueous humor by inhibiting the enzyme carbonic anhydrase.While such carbonic anhydrase inhibitors are now used to treatintraocular pressure by systemic and topical routes, current therapiesusing these agents, particularly those using systemic routes are stillnot without undesirable effects. Because carbonic anhydrase inhibitorshave a profound effect in altering basic physiological processes, theavoidance of a systemic route of administration serves to diminish, ifnot entirely eliminate, those side effects caused by inhibition ofcarbonic anhydrase such as metabolic acidosis, vomiting, numbness,tingling, general malaise and the like. Topically effective carbonicanhydrase inhibitors are disclosed in U.S. Pat. Nos. 4,386,098;4,416,890; 4,426,388; 4,668,697; 4,863,922; 4,797,413; 5,378,703,5,240,923 and 5,153,192.0

Prostaglandins and prostaglandin derivatives are also known to lowerintraocular pressure. U.S. Pat. No. 4,883,819 to Bito describes the useand synthesis of PGAs, PGBs and PGCs in reducing intraocular pressure.U.S. Pat. No. 4,824,857 to Goh et al. describes the use and synthesis ofPGD2 and derivatives thereof in lowering intraocular pressure includingderivatives wherein C-10 is replaced with nitrogen. U.S. Pat. No.5,001,153 to Ueno et al. describes the use and synthesis of13,14-dihydro-15-keto prostaglandins and prostaglandin derivatives tolower intraocular pressure. U.S. Pat. No. 4,599,353 describes the use ofeicosanoids and eicosanoid derivatives including prostaglandins andprostaglandin inhibitors in lowering intraocular pressure.

Prostaglandin and prostaglandin derivatives lower intraocular pressureby increasing uveoscleral outflow. This is true for both the F type andA type of Pgs and hence presumably also for the B, C, D, E and J typesof prostaglandins and derivatives thereof. A problem with usingprostaglandin derivatives to lower intraocular pressure is that thesecompounds often induce an initial increase in intraocular pressure, canchange the color of eye pigmentation and cause proliferation of sometissues surrounding the eye.

As can be seen, there are several current therapies for treatingglaucoma and elevated intraocular pressure, but the efficacy and theside effect profiles of these agents are not ideal. Recently potassiumchannel blockers were found to reduce intraocular pressure in the eyeand therefore provide yet one more approach to the treatment of ocularhypertension and the degenerative ocular conditions related thereto.Blockage of potassium channels can diminish fluid secretion, and undersome circumstances, increase smooth muscle contraction and would beexpected to lower IOP and have neuroprotective effects in the eye. (seeU.S. Pat. Nos. 5,573,758 and 5,925,342; Moore, et al., Invest.Ophthalmol. Vis. Sci 38, 1997; WO 89/10757, WO94/28900, and WO96/33719).

SUMMARY OF THE INVENTION

This invention relates to the use of potent potassium channel blockersor a formulation thereof in the treatment of glaucoma and otherconditions which are related to elevated intraocular pressure in the eyeof a patient. This invention also relates to the use of such compoundsto provide a neuroprotective effect to the eye of mammalian species,particularly humans. More particularly this invention relates to thetreatment of glaucoma and ocular hypertension (elevated intraocularpressure) using indole diterpene compounds having the structural formulaI:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method-for treating ocularhypertension or glaucoma which comprises administering to a patient inneed of such treatment a therapeutically effective amount of a compoundof Formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein,

-   R¹ is:    -   (a) H,-   R² is:    -   (a) CO₂C₁₋₆alkyl,    -   (b) H,    -   (c) OH, or-   R² and R⁷ are taken together to form an oxirane ring when b is a    single bond;-   R³ is:    -   (a) H, or    -   (b) (C═O)OC₁₋₆alkyl;-   R⁵ is:    -   (a) H,    -   (b) OH, or    -   (c) OC₁₋₆alkyl;-   R⁷ is H, OC₁₋₆ alkyl or R⁷ and R² are taken together to form an    oxirane ring when b is a single bond;-   Y is:    -   (a) H,    -   (b) OH,    -   (c) OC₁₋₆alkyl,    -   or Y and R⁸ are joined such that one of the following rings is        formed:-   R⁸ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl; and-   - - - is a double bond optionally present at a or b or at both a and    b.

Another embodiment of the invention is the method described abovewherein the compound of formula I is applied as a topical formulation.

Yet another embodiment is a method for treating ocular hypertension orglaucoma which comprises administering to a patient in need of suchtreatment a therapeutically effective amount of a compound selectedfrom:

and pharmaceutically acceptable salts, enantiomers, diastereomers andmixtures thereof.

Yet another embodiment contemplates the method described above whereinthe topical formulation is a solution or suspension.

And yet another embodiment is the method described above, whichcomprises administering a second active ingredient, concurrently orconsecutively, wherein the second active ingredient is selected from aβ-adrenergic blocking agent, a parasympathomimetic agent, a carbonicanhydrase inhibitor, and a prostaglandin or a prostaglandin derivative.

Another embodiment is the method described above wherein theβ-adrenergic blocking agent is timolol; the parasympathomimetic agent ispilocarpine; the carbonic anhydrase inhibitor is dorzolamide,acetazolamide, metazolamide or brinzolamide; the prostaglandin islatanoprost or rescula, and the prostaglandin derivative is ahypotensive lipid derived from PGF2α prostaglandins.

A further embodiment is a method for treating macular edema or maculardegeneration which comprises administering to a patient in need of suchtreatment a pharmaceutically effective amount of a compound ofstructural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein,

-   R¹ is:    -   (a) H,-   R² is:    -   (a) CO₂C₁₋₆alkyl,    -   (b) H,    -   (c) OH, or    -   R² and R⁷ are taken together to form an oxirane ring when b is a        single bond;-   R³ is:    -   (a) H, or    -   (b) (C═O)OC₁₋₆alkyl;-   R⁵ is:    -   (a) H,    -   (b) OH, or    -   (c) OC₁₋₆alkyl;-   R⁷ is H, OC₁₋₆ alkyl or R⁷ and R² are taken together to form an    oxirane ring when b is a single bond;-   Y is:    -   (a) H,    -   (b) OH,    -   (c) OC₁₋₆alkyl,    -   or Y and R⁸ are joined such that one of the following rings is        formed:-   R⁸ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl; and-   - - - is a double bond optionally present at a or b or at both a and    b.

Another embodiment is the method described above wherein the compound offormula I is applied as a topical formulation.

Yet another embodiment is a method for treating macular edema or maculardegeneration which comprises administering to a patient in need of suchtreatment a pharmaceutically effective amount of a compound selectedfrom:

and pharmaceutically acceptable salts, enantiomers, diastereomers andmixtures thereof.

A further embodiment is illustrated by a method for increasing retinaland optic nerve head blood velocity or increasing retinal and opticnerve oxygen tension which comprises administering to a patient in needof such treatment a therapeutically effective amount of a compound ofFormula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein,

-   R¹ is:    -   (a) H,-   R² is:    -   (a) CO₂C₁₋₆alkyl,    -   (b) H,    -   (c) OH, or    -   R² and R⁷ are taken together to form an oxirane ring when b is a        single bond;-   R³ is:    -   (a) H, or    -   (b) (C═O)OC₁₋₆alkyl;-   R⁵ is:    -   (a) H,    -   (b) OH, or    -   (c) OC₁₋₆alkyl;-   R⁷ is H, OC₁₋₆ alkyl or R⁷ and R² are taken together to form an    oxirane ring when b is a single bond;-   Y is:    -   (a) H,    -   (b) OH,    -   (c) OC₁₋₆alkyl,    -   or Y and R⁸ are joined such that one of the following rings is        formed:-   R⁸ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl; and-   - - - is a double bond optionally present at a or b or at both a and    b.

And another embodiment is the method described above wherein thecompound of formula I is applied as a topical formulation.

And still a further embodiment is a method for increasing retinal andoptic nerve head blood velocity or increasing retinal and optic nerveoxygen tension which comprises administering to a patient in need ofsuch treatment a therapeutically effective amount of a compound selectedfrom:

and pharmaceutically acceptable salts, enantiomers, diastereomers andmixtures thereof.

Another embodiment of the invention is a method for providing aneuroprotective effect to a mammalian eye which comprises administeringto a patient in need of such treatment a therapeutically effectiveamount of a compound of Formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein,

-   R¹ is:    -   (a) H,-   R² is:    -   (a) CO₂C₁₋₆alkyl,    -   (b) H,    -   (c) OH, or    -   R² and R⁷ are taken together to form an oxirane ring when b is a        single bond;-   R³ is:    -   (a) H, or    -   (b) (C═O)OC₁₋₆alkyl;-   R⁵ is:    -   (a) H,    -   (b) OH, or    -   (c) OC₁₋₆alkyl;-   R⁷ is H, OC₁₋₆ alkyl or R⁷ and R² are taken together to form an    oxirane ring when b is a single bond;-   Y is:    -   (a) H,    -   (b) OH,    -   (c) OC₁₋₆alkyl,    -   or Y and R⁸ are joined such that one of the following rings is        formed:-   R⁸ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl; and-   - - - is a double bond optionally present at a or b or at both a and    b.

Also within the scope of the invention is the method described abovewherein the compound of Formula I is applied as a topical formulation.

Another embodiment is represented by a method for providing aneuroprotective effect to a mammalian eye which comprises administeringto a patient in need of such treatment a therapeutically effectiveamount of a compound selected from:

and pharmaceutically acceptable salts, enantiomers, diastereomers andmixtures thereof.

Also contemplated to be within the scope of the present invention is thetopical formulation of Compound I as described above wherein the topicalformulation also contains xanthan gum or gellan gum.

The invention is described herein in detail using the terms definedbelow unless otherwise specified.

When any variable (e.g., aryl, alkyl, R¹, R², etc.) occurs more than onetime in any constituent or in a structural formula, its definition oneach occurrence is independent of its definition at every occurrence.Also, combinations of substituents and/or variables are permissible onlyif such combinations result in stable compounds.

Also included within the scope of this invention are pharmaceuticallyacceptable salts or esters, where a basic or acidic group is present ina compound of Formula I, such as, for example on the substituted alkylmoiety. When an acidic substituent is present, i.e. —COOH, there can beformed the ammonium, sodium, or calcium salt, and the like, for use asthe dosage form. Also, in the case of the —COOH group being present,pharmaceutically acceptable esters may be employed, e.g., acetate,maleate, pivaloyloxymethyl, and the like, and those esters known in theart for modifying solubility or hydrolysis characteristics for use assustained release or prodrug formulations.

Where a basic group is present, such as amino, acidic salts such ashydrochloride, hydrobromide, acetate, pamoate and the like may be usedas the dosage form.

As used herein “alkyl” is intended to include both branched- andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms and includes methyl (Me), ethyl (Et),propyl (Pr), butyl (Bu), pentyl, hexyl and the like. “Alkoxy” representsan alkyl group of the indicated number of carbon atoms attached throughan oxygen bridge. “Cycloalkyl” is intended to include saturated carbonring groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl(Cyh). “Alkenyl” is intended to include hydrocarbon groups of either astraight or branched configuration with one or more carbon-carbon doublebonds which may occur in any stable point along the chain, such asethenyl, propenyl, butenyl, and the like. “Alkynyl” is intended toinclude hydrocarbon groups of either a straight or branchedconfiguration with one or more carbon-carbon triple bonds which mayoccur in any stable point along the chain, such as ethynyl, propynyl,butynyl, and the like. “Halo” or “halogen” as used herein means fluoro,chloro, bromo and iodo. The term “Boc” refers to t-butyloxy-carbonyl.

This invention is also concerned with a method of treating ocularhypertension or glaucoma by administering to a patient in need thereofone of the compounds of formula I in combination with a β-adrenergicblocking agent such as timolol, a parasympathomimetic agent such aspilocarpine, carbonic anhydrase inhibitor such as dorzolamide,acetazolamide, metazolamide or brinzolamide, a prostaglandin such aslatanoprost, rescula, S1033 or a prostaglandin derivative such as ahypotensive lipid derived from PGF2α prostaglandins. An example of ahypotensive lipid (the carboxylic acid group on the α-chain link of thebasic prostaglandin structure is replaced with electrochemically neutralsubstituents) is that in which the carboxylic acid group is replacedwith a C₁₋₆ alkoxy group such as OCH₃ (PGF_(2a) 1-OCH₃), or a hydroxygroup (PGF_(2a) 1-OH).

Preferred potassium channel blockers are calcium activated potassiumchannel blockers. More preferred potassium channel blockers are highconductance, calcium activated potassium (maxi-K) channel blockers.Maxi-K channels are a family of ion channels that are prevalent inneuronal, smooth muscle and epithelial tissues and which are gated bymembrane potential and intracellular Ca²⁺.

Intraocular pressure (IOP) is controlled by aqueous humor dynamics.Aqueous humor is produced at the level of the non-pigmented ciliaryepithelium and is cleared primarily via outflow through the trabecularmeshwork. Aqueous humor inflow is controlled by ion transport processes.It is thought that maxi-K channels in non-pigmented ciliary epithelialcells indirectly control chloride secretion by two mechanisms; thesechannels maintain a hyperpolarized membrane potential (interiornegative) which provides a driving force for chloride efflux from thecell, and they also provide a counter ion (K⁺) for chloride ionmovement. Water moves passively with KCl allowing production of aqueoushumor. Inhibition of maxi-K channels in this tissue would diminishinflow. Maxi-K channels have also been shown to control thecontractility of certain smooth muscle tissues, and, in some cases,channel blockers can contract quiescent muscle, or increase the myogenicactivity of spontaneously active tissue. Contraction of ciliary musclewould open the trabecular meshwork and stimulate aqueous humor outflow,as occurs with pilocarpine. Therefore maxi-K channels could profoundlyinfluence aqueous humor dynamics in several ways; blocking this channelwould decrease IOP by affecting inflow or outflow processes or by acombination of affecting both inflow/outflow processes.

The present invention is based upon the finding that maxi-K channels, ifblocked, inhibit aqueous humor production by inhibiting net solute andH₂O efflux and therefore lower IOP. This finding suggests that maxi-Kchannel blockers are useful for treating other ophthamologicaldysfunctions such as macular edema and macular degeneration. It is knownthat lowering of IOP promotes increased blood flow to the retina andoptic nerve. Accordingly, this invention relates to a method fortreating macular edema, macular degeneration or a combination thereof.

Additionally, macular edema is swelling within the retina within thecritically important central visual zone at the posterior pole of theeye. An accumulation of fluid within the retina tends to detach theneural elements from one another and from their local blood supply,creating a dormancy of visual function in the area.

Glaucoma is characterized by progressive atrophy of the optic nerve andis frequently associated with elevated intraocular pressure (IOP). It ispossible to treat glaucoma, however, without necessarily affecting IOPby using drugs that impart a neuroprotective effect. See Arch.Ophthalmol. Vol. 112, Jan 1994, pp. 37-44; Investigative Ophthamol. &Visual Science, 32, 5, April 1991, pp. 1593-99. It is believed thatmaxi-K channel blockers which lower IOP are useful for providing aneuroprotective effect. They are also believed to be effective forincreasing retinal and optic nerve head blood velocity and increasingretinal and optic nerve oxygen by lowering IOP, which when coupledtogether benefits optic nerve health. As a result, this inventionfurther relates to a method for increasing retinal and optic nerve headblood velocity, increasing retinal and optic nerve oxygen tension aswell as providing a neuroprotective effect or a combination thereof.

The maxi-K channel blockers used are preferably administered in the formof ophthalmic pharmaceutical compositions adapted for topicaladministration to the eye such as solutions, ointments, creams or as asolid insert. Formulations of this compound may contain from 0.01 to 5%and especially 0.5 to 2% of medicament. Higher dosages as, for example,about 10% or lower dosages can be employed provided the dose iseffective in reducing intraocular pressure, treating glaucoma,increasing blood flow velocity or oxygen tension or providing aneuroprotective effect. For a single dose, from between 0.001 to 5.0 mg,preferably 0.005 to 2.0 mg, and especially 0.005 to 1.0 mg of thecompound can be applied to the human eye.

The pharmaceutical preparation which contains the compound may beconveniently admixed with a non-toxic pharmaceutical organic carrier, orwith a non-toxic pharmaceutical inorganic carrier. Typical ofpharmaceutically acceptable carriers are, for example, water, mixturesof water and water-miscible solvents such as lower alkanols oraralkanols, vegetable oils, polyalkylene glycols, petroleum based jelly,ethyl cellulose, ethyl oleate, carboxymethyl-cellulose,polyvinylpyrrolidone, isopropyl myristate and other conventionallyemployed acceptable carriers. The pharmaceutical preparation may alsocontain non-toxic auxiliary substances such as emulsifying, preserving,wetting agents, bodying agents and the like, as for example,polyethylene glycols 200, 300, 400 and 600, carbowaxes 1,000, 1,500,4,000, 6,000 and 10,000, antibacterial components such as quaternaryammonium compounds, phenylmercuric salts known to have cold sterilizingproperties and which are non-injurious in use, thimerosal, methyl andpropyl paraben, benzyl alcohol, phenyl ethanol, buffering ingredientssuch as sodium borate, sodium acetates, gluconate buffers, and otherconventional ingredients such as sorbitan monolaurate, triethanolamine,oleate, polyoxyethylene sorbitan monopalmitylate, dioctyl sodiumsulfosuccinate, monothioglycerol, thiosorbitol, ethylenediaminetetracetic acid, and the like. Additionally, suitable ophthalmicvehicles can be used as carrier media for the present purpose includingconventional phosphate buffer vehicle systems, isotonic boric acidvehicles, isotonic sodium chloride vehicles, isotonic sodium boratevehicles and the like. The pharmaceutical preparation may also be in theform of a microparticle formulation. The pharmaceutical preparation mayalso be in the form of a solid insert. For example, one may use a solidwater soluble polymer as the carrier for the medicament. The polymerused to form the insert may be any water soluble non-toxic polymer, forexample, cellulose derivatives such as methylcellulose, sodiumcarboxymethyl cellulose, (hydroxyloweralkyl cellulose), hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose;acrylates such as polyacrylic acid salts, ethylacrylates,polyactylamides; natural products such as gelatin, alginates, pectins,tragacanth, karaya, chondrus, agar, acacia; the starch derivatives suchas starch acetate, hydroxymethyl starch ethers, hydroxypropyl starch, aswell as other synthetic derivatives such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, neutralizedcarbopol and xanthan gum, gellan gum, and mixtures of said polymer.

Suitable subjects for the administration of the formulation of thepresent invention include primates, man and other animals, particularlyman and domesticated animals such as cats and dogs.

The pharmaceutical preparation may contain non-toxic auxiliarysubstances such as antibacterial components which are non-injurious inuse, for example, thimerosal, benzalkonium chloride, methyl and propylparaben, benzyldodecinium bromide, benzyl alcohol, or phenylethanol;buffering ingredients such as sodium chloride, sodium borate, sodiumacetate, sodium citrate, or gluconate buffers; and other conventionalingredients such as sorbitan monolaurate, triethanolamine,polyoxyethylene sorbitan monopalmitylate, ethylenediamine tetraaceticacid, and the like.

The ophthalmic solution or suspension may be administered as often asnecessary to maintain an acceptable IOP level in the eye. It iscontemplated that administration to the mammalian eye will be about onceor twice daily.

For topical ocular administration the novel formulations of thisinvention may take the form of solutions, gels, ointments, suspensionsor solid inserts, formulated so that a unit dosage comprises atherapeutically effective amount of the active component or somemultiple thereof in the case of a combination therapy.

The maxi-K channel blockers used in the present invention are made by amicrobiological processes employing the Culture Nalanthalama sp. (MF5785). The process has been previously described in U.S. Pat. No.5,541,208, herein incorporated by reference. This culture, ATCC 74192,is available from the American Type Culture Collection located at 12301Parklawn Drive in Rockville, Md. Modified isoxazolines, such as alkylesters, can be made by modifications of the fungal isolates viasynthetic protocols known in the art.

The starting material for the fermentation may be selected frompaxilline or any indole diterpene having a hydroxyl group beta to acarbonyl group. The diterpene alkaloid of the formula:

is known in the art as Paxilline and is produced from a number ofmicroorganisms. Paxilline, and the related compounds paspalitrem andaflatrem are fungal metabolites known to be tremorgens. Paxilline or arelated indole diterpene containing the necessary hydroxyl-carbonylfunctionality may be reacted with hydroxylamine in a suitable solventunder appropriate conditions to form the oxime which is further reactedwith tributylphosphine and diphenyldisulfide to produce an isoxazolinesuch as paxizoline. The claimed process is not limited to producingpotassium channel antagonists such as paxizoline but may also be used toproduce pharmaceutically active isoxazolines or drugs containing anisoxazoline ring. The scheme below indicates the required functionalityand describes the general process:

As shown above, the claimed process relates generally to the initialformation of an oxime which subsequently reacts with tributylphosphineand diphenyldisulfide to form the heterocycle isoxazoline or isoxazole.

The free acid groups on the compounds produced by the microbiologicalprocess may also be readily modified to the alkyl ester. For example,trimethylsilyldiazomethane added to a methanolic solution containing afree acid moiety is readily converted to the methyl ester. Other simplealkyl or aryl or benzyl esters may readily be prepared by conventionalmeans to produce compounds within the scope of the present invention.

The indole nitrogen may readily be protected with a suitable protectinggroup selected from, for example, t-butyloxycarbonyl or other protectinggroup selected from groups described in “Protective Groups in OrganicSynthesis” by Greene and Wutts (1991). Furthermore, the free alcoholmoieties may also be protected using standard hydroxyl protectinggroups.

The compounds used in the present invention can be made by afermentation process for producing potassium channel antagonistscomprising:

(a) inoculating seed medium (Table 1) with mycelia of Nalanthalama sp.MF5785 (ATCC 74192);

(b) incubating the inoculated mycelia at room temperature (20-30° C.)under humid conditions with constant fluorescent light, preferably withshaking, most preferably on a rotary shaker with a 5-cm throw at 220rpm;

(c) using the culture produced in step (b) to inoculate a liquidproduction medium and further incubating under the conditions defined instep (b) to produce Compounds A, B, C, D and G.

Maximal accumulation of compounds A, B, C, D and G in the fermentationbroth occurs between 7-11 days. The invention further comprises a step(d) in which the compounds produced in the fermentation broth undersuitable defined and controlled conditions are purified and isolatedfrom the broth. Suitable isolation procedures include, for example,extraction of the culture medium with an alcoholic or oxygenatedsolvent, such as an ether or ketone, preferably methylethylketone.

The strain MF5785 has been identified as an Nalanthaloma. The fungus wasisolated from unidentified twigs collected in the province of NuevoLeon, Mexico. The generic disposition is based the undifferentiated,unbranched, solitary, enteroblastic, phialidic conidiogenous cells thatgive rise to conidia that are small, subglobose, hyaline and smooth.Within the genus Acremonium, this isolate could be assigned to theseries terricola because the conidia adhere to the conidogenous cells indry chains. However, the isolate does not match any of the taxa in thisseries presented by Gams in his monograph of the genus Acremonium (W.Grams. 1971. Cephalosporium-artige Schimmelpilze (Hyphomycetes). Thisorganism grows well and sporulates abundantly in most mycological media.In agar culture, the strain exhibits the following morphologicalfeatures:

Colonies growing moderately well on oatmeal agar (Difco Laboratories),25° C., 12 hr photoperiod, after 21 days attaining 34-36 mm in diameter,slightly raised, velvety, finely cottony, becoming minutely granular atthe center, dull, dry, faintly zonateith margin even and submerged,white at the margin becoming dull vinaceous to grayish vinaceous, PaleVinaceous-Fawn, Vinaceous-Buff, Light Vinaceous-Fawn, to Vinaceous-Fawn(capitalized color names from Ridgway, R. 1912. Color Standards andNomenclature, Washington, D.C.). Odors and exudates absent.

Colonies growing moderately fast on Emerson Yp Ss (Difco Laboratories)agar, 25° C., 12 hr photoperiod, after 21 days attaining 30 mm diameter,appressed to slightly raised, faintly radially sulcate, faintly zonate,velvety, with minute drops of watery exudate towards center, dry, dull,with margin submerged, minute fimbriate to wavy, translucent, white topale vinaceous, Pale Vinaceous-Fawn, Light Vinaceous-Fawn, translucentto pale yellow in reverse. Odors and exudates absent.

Colonies growing moderately fast on Barnett's oak wilt agar (Barnett, H.L. 1953. Isolation and identification of the oak wilt fungus. WestVirginia Agricultural Experiment Station Bulletin 359T: 1-15.), 25° C.,12 hr photoperiod, after 21 days attaining 25 mm diameter, appressed,pruinose to downy, farinaceous towards the center, radially rivulose,faintly zonate, with margin even and submerged, white to pale vinaceous,with farinaceous granules pale vinaceous gray. Granular texture causedby aggregation of conidiophores into small pustules and accumulations ofdry conidia. Odors and exudates absent.

No growth occured at 37° C. on Emerson Yp Ss agar after 21 days.

Conidiophores micronematous, occasionally semi-micronematous,integrated, up to 30 μm tall, but usually 6-12 μm tall, branched or not,septate or not, often only a simple right-angle branch from main hyphalaxis, usually with a single terminal conidiogenous locus, butoccasionally conidiogenous loci are lateral or intercalary.Conidiogenous cells terminal or intercalary, appearing enteroblastic andphialidic, occasionally swollen at the base. Conidia hyaline,thin-walled, broadly ellipsoidal or obovate, with a slightly flattenedbase, 2-5×15.2.5 μm, accumulating in dry chains, sometime with faintconnectives evident.

Hyphae septate, branched, finely incrusted in mature regions of thecolonies.

In general, Compounds A,B,C, D and G can be produced by culturing(fermenting) strain MF5785, ATCC 74192, in an aqueous nutrient mediumcontaining assimilable carbon and nitrogen sources, preferably undersubmerged aerobic conditions, and shaking the culture under constantfluorescent light, preferably 450 to 700 nm, until a substantial amountof Compounds A,B,C, D and G is detected in the fermentation broth. Theculture is incubated in a aqueous medium at a temperature between 20° C.and 37° C., preferably 25° C. for a period of time necessary to completethe formation of Compounds A,B,C, D, and G, usually for a period between3 to 28 days, preferably between 7 to 11 days, preferably on a shakingmeans, most preferably on a rotary shaker operating at 220 rpm with a 5cm throw. The aqueous production medium is maintained at a pH between 5and 8, preferably about 6.0, at the initiation and termination (harvest)of the fermentation process. The desired pH may be maintained by the useof a buffer such as [2-(N-morpholino)ethanesulfonic acid]monohydrate(MES), 3-(N-morpholino)propanesulfonic acid (MOPS), phosphate buffer orany other buffer effective in pH 5 to 8, or by choice of nutrientmaterials which inherently possess buffering properties, such asproduction media described herein below. The active compound isextracted from the mycelial growth of the culture is with a suitablesolvent, such as alcoholic or oxygenated solvent such as an ester orketone. The preferred solvent for extraction is methylethylketone (MEK).The solution containing the desired compound is concentrated and thensubjected to chromatographic separation to isolate compounds A,B,C, Dand G from the cultivation medium.

The preferred sources of carbon in the nutrient medium include sucrose,glucose, fructose, mannitol, glycerol, xylose, galactose, lactose,sorbitol, starch, dextrin, other sugars and sugar alcohols, starches andother carbohydrates, or carbohydrates derivatives, and the like. Othersources which may be included are maltose, rhamnose, raffinose,arabinose, mannose, salicin, sodium succinate, acetate, and the like aswell as complex nutrients such as yellow corn meal, oat flour, millet,rice, cracked corn, and the like. The exact quantity of the carbonsource which is utilized in the medium will depend, in part, upon theother ingredients in the medium, but it is usually found that an amountof carbohydrate between 0.5 and 15 percent by weight of the medium issatisfactory. These carbon sources can be used individually or severalsuch carbon sources may be combined in the same medium.

The preferred sources of nitrogen are yeast extract, yellow corn meal,meat extract, peptone, gluten meal, cottonseed meal, soybean meal andother vegetable meals (partially or totally defatted), caseinhydrolysates, soybean hydrolysates and yeast hydrolysates, corn steepliquor, dried yeast, wheat germ, feather meal, peanut powder,distiller's solubles, etc., as well as inorganic and organic nitrogencompounds such as ammonium salts (e.g. ammonium nitrate, ammoniumsulfate, ammonium phosphate, etc.), urea, amino acids such asmethionine, phenylalanine, serine, alanine, proline, glycine, arginineor threonine, and the like. The various sources of nitrogen can be usedalone or in combination in amounts ranging from 0.2 to 10 percent byweight of the medium.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form because less purematerials which contain traces of growth factors and considerablequantities of mineral nutrients are also suitable for use. When desired,there may be added to the medium inorganic salts, sodium, potassium,magnesium, calcium, phosphate, sulfate, chloride, carbonate, and likeions which can be incorporated in the culture medium as sodium orcalcium carbonate, sodium or potassium phosphate, sodium or potassiumchloride, sodium or potassium iodide, magnesium salts, copper salts,cobalt salts, and the like. Also included are trace metals such ascobalt, manganese, iron, molybdenum, zinc, cadmium, copper, and thelike. The various sources of inorganic salts can be used alone or incombination in amounts ranging from 0.1 to 1.0 , and trace elementsranging from 0.001 to 0.1 percent by weight of the medium.

If necessary, especially when the culture medium foams seriously, adefoaming agent, such as polypropylene glycol 2000 (PPG-2000), liquidparaffin, fatty oil, plant oil, mineral oil or silicone may be added.

Submerged aerobic fermentation conditions in fermentors are preferredfor the production of Compounds A,B,C, D and G in large amounts. For theproduction in small amounts, a shaking or surface culture in a flask orbottle is employed. Furthermore, when the growth is carried out in largetanks, it is preferable to use the vegetative form of the organism forinoculation in the production tanks in order to avoid growth lag in theprocess of production of Compounds A,B,C, D and G. Accordingly, it isdesirable first to produce a vegetative inoculum of the organism byinoculating a relatively small quantity of culture medium with spores ormycelia of the organism produced in a “slant,” or from previouslyprepared frozen mycelia, and culturing the inoculated medium, alsocalled the “seed medium”, and then aseptically transferring the culturedvegetative inoculum to large tanks. The seed medium, in which theinoculum is produced may be seen in Table 1 and is generally autoclavedto sterilize the medium prior to inoculation. The seed medium isgenerally adjusted to a pH between 5 and 8, preferably about 6.8, priorto the autoclaving step by suitable addition of an acid or base,preferably in the form of a dilute solution of hydrochloric acid orsodium hydroxide. Growth of the culture in this seed medium ismaintained between 26° C. and 37° C., preferably 25° C. Incubation ofculture MF5785 (ATCC 74192) in a seed medium, preferably that in Table1, is usually conducted for a period of about 2 to 6 days, preferably 3to 4 days, with shaking, preferably on a rotary shaker operating at 220rpm with a 5 cm throw; the length of incubation time may be variedaccording to fermentation conditions and scales. If appropriate, asecond stage seed fermentation may be carried out in the seed medium(Table 1) for greater production of mycelial mass by inoculating freshseed medium with a portion of the culture growth and then incubatingunder similar conditions but for a shortened period. The resultinggrowth then may be employed to inoculate, a production medium,preferably the Liquid Production Medium (Table 2). The fermentationliquid production medium inoculated with the seed culture growth isincubated for 3 to 28 days, usually 7 to 11 days, with agitation.Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentationmixture within the fermentor, by various pumping equipment or by thepassage of sterile air through the medium. Aeration may be effected bypassing sterile air through the fermentation mixture.

Preferred seed and production media for carrying out the fermentationinclude the following media:

TABLE 1 Seed Medium Trace Element Mix per liter per liter Corn SteepLiquor 5 g FeSO₄.H₂O 1 g Tomato Paste 40 g MnSO₄.4H₂O 1 g Oat flour 10 gCuCl₂.2H₂O 25 mg Glucose 10 g CaCl₂ 100 mg Trace element mix 10 mL H₃BO₃56 mg (NH₄)₆Mo₇O₂₄.4H₂O 19 mg pH = 6.8 ZnSO₄.7H₂O 200 mg

TABLE 2 Liquid Production Medium Component Per Liter Yellow Cornmeal50.0 g Yeast Extract 1.0 g Sucrose 80.0 g Distilled Water 1000.0 mL

The following examples are provided to illustrate the present inventionand should not be construed as limiting the scope of the invention.

EXAMPLE 1

Production of Compounds A, B, C, D and G

Step A: Fermentation Conditions for Production of Compounds A-D and G

Fermentation conditions for the production of Compounds A, B, C, D and Gby the microorganism Nalanthamala sp. were as follows: vegetativemycelia of a culture of the above microorganism were prepared byinoculating 54 mL of seed medium (Table 1) in a 250 mL unbaffledErlenmeyer flask with frozen mycelia of MF5785 (ATCC 74192). Seed flaskswere incubated at 25° C. and 50% relative humidity on a rotary shakerwith a 5-cm throw at 220 rpm in a room with constant fluorescent light,about 400 to 750 nm. Two-mL portions of the resulting 3-day culture wereused to inoculate 50 mL portions of Liquid Production Medium (Table 2)in 250 mL unbaffled Erlenmeyer flasks; these cultures were incubated at25° C., 220 rpm with 50% relative humidity in a room with constantfluorescent light. The products appeared in the fermentation as early as7 days with maximal accumulation observed at day 11. At harvest, thecompounds were extracted as described below.

Step B: Isolation of Compounds A,B,C, D and G

The fermentation broth of MF5785 (ATCC 74192), prepared above (3 L WBE;IC₅₀=10 μl WBE per mL in [¹²⁵I]ChTX binding assay (described in Example5) was extracted with methyl ethyl ketone, and the solvent was removedin vacuo. The dry residue was then partitioned between CH₂Cl₂ and H₂O togive 4.5 g in organic phase. The aqueous phase was treated with methanolafter the water was removed to yield 1.6 g. The organic phases weresubjected to flash chromatography on SiO₂ using CH₂Cl₂-CH₃OH to resultin two fractions, I (2.36 g) and II (1.97 g). Reverse phase flashchromatography on BAKERBOND C₁₈ using methanol-water on each fractionwas followed by HPLC on PARTISIL 10 ODS-3 (22×50; flow rate 10 mL permin).

The fraction I yielded several compounds in different amounts uponpurification by HPLC (70% CH₃CN—H₂O) as follows:

Compound A (C₃₂H₄₃NO₃, M.W. 489.3243 (calcd), 489.3226 (found)) of thechemical formula:

Compound B (C₃₇H₅₁NO₅, M.W. 589.3767 (calcd), 589.3749 (found)) of thechemical formula:

Compound C (C₃₇H₄₇NO₅, M.W. 585.3506 (calcd), 585.3454 (found)) of thechemical formula:

The fraction II provided hydroxypaspalinic acid (Compound D) upon HPLC(50% CH₃OH—H₂O) and Compound G:

Compound D (29.2 mg; C₂₈H₃₇NO₅; M.W. 467.2672 (calcd), 467.2641 (found))of the chemical formula:

Compound G (C₃₇H₄₉NO₆; M.W. 603.3559 (calcd))

¹³C NMR Data

¹³C NMR spectra were recorded in CD₂Cl₂ and CD₃OD at 125 MHz on VarianUnity 500 NMR spectrometer at 25° C. Chemical shifts are given in ppmrelative to tetramethylsilane (TMS) at zero ppm using the solvent peakat 53.8 (CD₂Cl₂) and 49.0 (CD₃OD) ppm as internal standard.

Compound A (CD₂Cl₂): 16.0, 17.9, 18.0, 18.7, 19.2, 21.0, 23.7, 25.77,25.81, 27.5, 27.7, 28.1, 30.8, 34.8, 42.7, 50.4, 51.0, 58.4, 64.9, 67.5,69.1, 71.8, 75.3, 78.2, 79.5, 111.1, 117.6, 118.4, 120.8, 121.2, 123.7,124.6, 132.0, 134.6, 137.4, 140.6, 151.8 ppm. The carbon count of 37 isin agreement with the HR-EIMS derived molecular formula C₃₇H₅₁NO₅.

Compound B (CD₂Cl₂): 14.6, 16.6, 18.0, 21.3, 22.4, 23.3, 24.5, 25.8,26.6, 27.5, 29.7, 32.9, 39.8, 41.3, 50.0, 50.7, 56.2, 59.1, 61.8, 73.89,73.97, 76.0, 111.7, 118.5, 118.6, 119.8, 120.7, 122.9, 125.4, 135.1,140.3, 150.5 ppm. The carbon count of 32 is in agreement with theHR-EIMS derived molecular formula C₃₂H₄₃NO₃.

Compound C (CD₂Cl₂): 16.2, 16.8, 18.0, 18.7, 20.1, 20.8, 25.6, 25.8,28.1, 29.4, 30.8 (2×), 32.3, 44.1, 50.4, 50.7, 60.7, 65.2, 71.4, 73.9,75.0, 76.6, 93.2, 106.3, 109.5, 117.2, 119.3, 121.2, 122.2, 124.2,124.9, 132.1, 133.4, 139.8, 140.2, 144.9, 151.3 ppm. The carbon count of37 is in agreement with the HR-EIMS derived molecular formula C₃₇H₄₇NO₅.

Compound D (CD₃OD): 14.8, 16.9, 24.7, 25.4, 26.1, 26.2, 26.3, 28.3,32.3, 33.9, 40.8, 41.5, 50.3, 53.5, 53.7, 68.3, 72.6, 77.6, 79.7, 112.6,118.0, 118.7, 119.7, 120.7, 126.2, 142.1, 152.1, 178.1 ppm. The carboncount of 28 is in agreement with the HREI-MS derived molecular formulaC₂₈H₃₇NO₅.

Compound G (CD₂Cl₂): 16.0, 16.8, 18.7, 18.9, 19.1, 20.9, 25.0, 25.6,27.7, 28.35, 28.44, 29.4, 30.7, 33.1, 42.7, 50.6, 50.7, 58.8, 61.5,64.6, 68.2, 71.4, 71.7, 72.0, 74.9, 78.5, 93.0, 110.2, 117.0, 119.7,121.1, 122.6, 125.1, 129.6, 139.3, 140.0, 151.9 ppm. The NMR dataindicates a carbon count of 37 and a molecular formula C₃₇H₄₇NO₆.¹H NMR Data

¹H NMR spectra were recorded at 500 MHz in CD₂Cl₂ Varian XL300 on aUnity 500 NMR spectrometer at 25° C. and in CD₃OD for Compound D at 300MHz on a Varian XL300 spectrometer at ambient temperature. Chemicalshifts are indicated in ppm relative to TMS at zero ppm using thesolvent peak at δ 5.32 (CD₂Cl₂) and 3.30 (CD₃OD) as internal standard.Only diagnostic peaks are noted.

Compound A (CD₂Cl₂): δ 1.11 (3H, s), 1.22 (3H, s), 1.25 (3H, s), 1.28(3H, s), 1.66 (3H, br s), 1.73 (3H, br s), 1.74 (6H, br s), 1.91 (1H,m), 2.03 (1H, m), 2.26 (1H, m), 2.36 (1H dd, J=11, 13 Hz), 2.66 (1H, dd,J=6.5, 13 Hz), 2.79 (1H, m), 3.34 (1H, d, J=9 Hz), 3.38 (2H, m), 3.56(1H, br s), 3.96 (3H, m), 4.17 (1H, t, J=8.5 Hz), 5.26 (1H, m), 5.35(1H, m), 6.86 (1H, dd, J=1.5, 8 Hz), 7.09 (1H br s), 7.17 (1H, d, J=8Hz), 7.74 (1H, br s, NH).

Compound B (CD₂Cl₂): δ 1.02 (3H, s), 1.08 (3H, s), 1.11 (3H, s), 1.14(3H, s), 1.64 (3H, br s), 1.72 (3H, br s), 1.86 (1H, m), 1.94 (1H, m),2.09 (1H, m), 2.25 (1H, m), 2.37 (1H, dd, J=10.5, 13.5 Hz), 2.68 (1H,dd, J=6.5, 13.5 Hz), 2.79 (1H, m), 3.42 (1H, br d, J=3 Hz), 3.66 (1H,dd, J=2.5, 11 Hz), 3.88 (1H, m) 3.96 (1H, m), 5.25 (1H, m), 5.35 (1H,m), 7.02 (1H, dt, J=1.5, 7 Hz), 7.05 (1H, dt, J=1.5, 7 ), 7.30 (1H, m),7.39 (1H, m), 7.88 (1H, br s, NH).

Compound C (CD₂Cl₂): δ 1.17 (3H, s), 1.30 (3H, s), 1.31 (3H, s), 1.32(3H, s), 1.71 (3H, d, J=1.5 Hz), 1.74 (6H, d, J˜1 Hz), 1.76 (3H, d, J˜1Hz), 1.67 (1H, m), 1.88 (1H, dd, J=7, 16 Hz), 1.95 (1H, m), 2.57 (1H,dd, J=10.5, 13.0 Hz), 2.77 (1H, m), 2.84 (1H, dd, J=6.5, 13.0 Hz), 3.18(1H, br d, J˜16 Hz), 3.58 (1H, br d, J=7 Hz), 3.86 (1H, s), 3.96 (1H, d,J=10 Hz), 4.00 (1H, d, J=10 Hz), 5.21 (1H, m), 5.35 (1H, m), 5.39 (1H,dd, J=2, 7 Hz), 5.47 (1H, d, J=6.5 Hz), 6.81 (1H, dd, J=1, 7 Hz), 6.96(1H, dd, J=7, 8 Hz ),7.14 (1H, dd, J=1, 8 Hz), 7.83 (1H, br s, NH).

Compound D (CD₃OD at 300 MHz): δ 1.01 (3H, s), 1.09 (3H, s), 1.12 (3H,s), 1.20 (3H, s), 2.21 (1H,dd, J=4, 12 Hz), 2.29 (1H, dd, J=10.5, 13.0Hz), 2.61 (1H, dd, J=6.5, 13.0 Hz), ˜2.73 (1H, m), 3.64 (1H, dd, J=3, 11Hz), 3.67 (1H, dd, J=4.5, 12 Hz), 4.37 (1H, t, J=3 Hz), 6.92 (2H, m),7.27 (2H, m).

Compound G (CD₂Cl₂): d 1.15 (3H, s), 1.24 (3H, s), 1.27 (3H, s), 1.28(3H, s), 1.31 (3H, s), 1.40 (3H, s), 1.71 (3H, d, J=1 Hz), 1.74 (3H, d,J=1 Hz), 1.81 (1H, m), 1.93 (1H, m), 2.27 (1H, m), 2.59 (1H, dd, J=13,15 Hz), 2.69 (1H, dt, J=5, 13.5 Hz), 2.84 (2H, m), 3.14 (2H, m), 3.16(1H, m), 3.49 (1H, d, J=9.5 Hz), 3.59 (1H, s), 3.91 (1H, dd, J=1, 9.5Hz), 4.32 (1H, br t, J˜9 Hz), 5.20 (1H, m), 5.50 (1H, d, J=6.5 Hz), 6.87(1H, dd, J=1, 7.5 Hz), 7.00 (1H, dd, J=7.5, 8 Hz ), 7.19 (1H, dd, J=1, 8Hz), 7.93 (1H, br s, NH).

Abbreviations: s=singlet, d=doublet, q=quartet, br=broad, m=multiplet,J=¹H—¹H coupling constant in Hertz (⁺0.5 Hz).

EXAMPLE 2

Preparation of Methyl Hydroxypaspalinate, Compound E

To a methanolic solution of the hydroxypaspalinic acid (Compound D, 7.8mg, 0.016 mM) was added (trimethylsilyl)diazomethane ((CH₃)₃SiCHN₂, 2 Msolution in hexanes) in excess. The mixture was stirred at roomtemperature until the faint yellow color dissipated. The solvent wasthen removed under nitrogen to give the methyl ester (TLC 5 %CH₃OH—CH₂Cl₂, R_(f) 0.19 (acid), 0.39 (ester)). It was purified by HPLCon PARTISIL 10 ODS-3 (22×50) using 60% CH₃CN-H₂O (flow rate 10 mL permin) to afford methyl hydroxypaspalinate (Compound E; M.W. 481). It waseluted at 149.6 min. and has the following formula:

¹³C NMR spectrum was recorded in CD₃OD at 75 MHz on a Varian XL300 NMRspectrometer at ambient temperature. Chemical shifts are given in ppmrelative to tetramethylsilane (TMS) at zero ppm using the solvent peakat 49.0 (CD₃OD) ppm as internal standard. 14.8, 16.6, 24.6, 25.4, 25.9,26.0, 26.2, 28.3, 32.5, 33.7, 40.6, 41.6, 50.3, 51.5, 53.7, 53.8, 68.1,72.6, 77.1, 79.4, 112.6, 118.0, 118.7, 119.7, 120.7, 126.2, 142.1,151.9, 176.2 ppm. The carbon count of 29 and chemical shift positionsare consistent with its assignment as the methyl ester of Compound D.

¹H NMR spectrum was recorded at 300 MHz in CD₃OD on a Varian XL300spectrometer at ambient temperature. Chemical shifts are indicated inppm relative to TMS at zero ppm using the solvent peak at δ3.30 (CD₃OD)as internal standard. Only diagnostic peaks are noted. δ 0.96 (3H, s),1.00 (3H, s), 1.12 (3H, s), 1.16 (3H, s), 1.42 (1H, ddd, J=2.5, 12, 14Hz), 1.91 (2H, m), 2.22 (1H, m), 2.28 (1H, dd, J=10.5, 13.0 Hz), ˜2.59(1H, m), 2.60 (1H, dd, J=6.5, 13.0 Hz), ˜2.69 (1H, m), 3.62 (1H, dd,J=2, 12 Hz), 3.65 (3H, s), 3.68 (1H, dd, J=4.0, 12 Hz), 4.35 (1H, t,J=˜3 Hz), 6.92 (2H, m), 7.26 (2H, m).

EXAMPLE 3

Synthesis of Paxizoline, Compound F

Crystalline NH₂OH.HCl (32 mg, 0.45 mM) was added to a solution ofpaxilline (20 mg, 0.045 mM) in C₂H₅OH (2 mL). The mixture was flushedwith nitrogen and stirred until the reaction, monitored by TLC (SiO₂,10% CH₃OH—CH₂Cl₂; R_(f) 0.67 (ketone) and 0.56 (oxime)), was complete.The solvent was removed, then the residue was transferred to aseparatory funnel using ether (5 mL). The organic layer was washedconsecutively with water (3×1 mL), saturated aqueous NaCl (1×1 mL), thendried with anhydrous MgSO₄, filtered through a sintered glass. Thesolvent was removed in vacuo. The crude mixture was purified by TLC(SiO₂ 60 F-254, 5% CH₃OH—CH₂Cl₂) to give paxilline oxime (18.6 mg;C₂₇H₃₄O₄N₂, M.W. 450.2518 (calcd), 450.2517 (found)).

To a mixture of paxilline oxime (10 mg, 0.022 mM) and diphenyl disulfide(4.85 mg, 0.022 mM) in dry tetrahydrofuran (2 mL) was addedtributylphosphine (9 mg, 0.044 mM). The mixture was stirred at roomtemperature overnight under nitrogen, then the solvent was removed. Thecrude product was purified by HPLC on PARTISIL 10 ODS-3 (9.4×50) using70% CH₃OH—H₂O (flow rate 3 mL per min) to give Compound F; 7.5 mg,C₂₇H₃₂N₂O₃, M.W. 432.2413 (calcd), 432.2403 (found), UV (CH₃OH) λ_(max)(nm) 234, 263, λ_(min) 252).

¹³C NMR(CD₂Cl₂) 75 MHz on Varian XL300 NMR spectrometer at ambienttemperature. Chemical shifts are given in ppm relative totetramethylsilane (TMS) at zero ppm using the solvent peak at 53.8(CD₂Cl₂) ppm as internal standard: 16.3, 19.9, 20.3, 21.3, 25.3, 27.4,28.5, 28.8, 35.1, 43.3, 50.0, 51.1, 74.9, 78.3, 85.8, 86.2, 109.0,111.7, 117.5, 118.6, 119.8, 120.7, 125.5, 140.1, 152.4, 154.6, 155.2ppm. The NMR data indicates a carbon count of 27 consistent with themolecular formula C₂₇H₃₂N₂O₃.

¹H NMR spectrum was recorded at 300 MHz in CD₂Cl₂ on a Varian XL300spectrometer at ambient temperature. Chemical shifts are indicated inppm relative to TMS at zero ppm using the solvent peak at δ5.32 (CD₂Cl₂)as internal standard. Only diagnostic peaks are noted. δ 1.02 (3H, s),1.20 (3H, s), 1.33 (3H, s), 1.53 (3H, s), 1.90 (1H, m), 2.24 (1H, m),2.43 (1H, dd, J=11, 13 Hz), 2.73 (1H, dd, J=6.5, 13 Hz), ˜2.74 (1H, m),2.86 (1H, m), 4.54 (1H, s), 4.87 (1H, ddd, J=2.5, 7, 10 Hz), 6.31 (1H,d, J=2 Hz), 7.03 (2H, m), 7.30 (1H, m), 7.40 (1H, m), 7.86 (1H, br s,NH).

EXAMPLE 4

Electrophysiological Experiments

Methods:

Patch clamp recordings of currents flowing through large-conductancecalcium-activated potassium (maxi-K) channels were made from membranepatches excised from cultured bovine aortic smooth muscle cells usingconventional techniques (Hamill et al., 1981, Pflügers Archiv. 391,85-100) at room temperature. Glass capillary tubing (Garner #7052) waspulled in two stages to yield micropipettes with tip diameters ofapproximately 1-2 microns. Pipettes were typically filled with solutionscontaining (mM): 150 KCl, 10 Hepes (4-(2-hydroxyethyl)-1-piperazinemethanesulfonic acid), 1 Mg, 0.01 Ca, and adjusted to pH 7.20 with 3.7mM KOH. After forming a high resistance (>10⁹ ohms) seal between thesarcolemmal membrane and the pipette, the pipette was withdrawn from thecell, forming an excised inside-out membrane patch. The patch wasexcised into a bath solution containing (mM): 150 KCl, 10 Hepes, 5 EGTA(ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid),sufficient Ca to yield a free Ca concentration of 1-5 μM, and the pH wasadjusted to 7.2 with 10.5 KOH. For example, 4.568 mM Ca was added togive a free concentration of 2 μM at 22° C. An AXOPATCH 1C amplifier(Axon Instruments, Foster City, Calif.) with a CV-4 headstage was usedto control the voltage and to measure the currents flowing across themembrane patch. The input to the headstage was connected to the pipettesolution with a Ag/AgCl wire, and the amplifier ground was connected tothe bath solution with a Ag/AgCl wire covered with a tube filled withagar dissolved in 0.2 M KCl. Maxi-K channels were identified by theirlarge single channel conductance (˜250 pS) and sensitivity of channelopen probability to membrane potential and intracellular calciumconcentration.

Data were stored on a RACAL STORE 4DS FM tape recorder (Racal Recorders,Vienna, Va.) or on digital video tape using a video casette recorderafter digitizing the signal with VR-10 (Instrutech Corp., Belmont, N.Y.)PCM video encoder. The signal was also recorded on chart paper with aGOULD 2400S chart recorder (Gould Inc., Cleveland, Ohio). Forquantitative analysis, the data was played into a DEC 11-73 (DigitalEquipment Corp., Maynard, Mass.) after digitization with a DT2782-8D1Aanalogue to digital converter (Data Translation Inc., Marlboro, Mass.),or played into a Mac IIx or Quadra 700 computer (Apple Computers) afterdigitization with an ITC-16 interface (Instrutech Corp., Belmont, N.Y.).

Results:

The effects of the compounds of the present invention on maxi-K channelsfrom bovine aortic smooth muscle were examined in excised inside-outmembrane patches. Addition of 10 nM Compound C to the bath produced arapid and complete block of maxi-K channels that was not reversed duringa brief (˜10 min) washout. 1 nM of Compound B caused nearly completeblock of maxi-K channels suggesting a K_(i) of less than 1 nM forchannel block. Compound D, Compound A and Compound E were weakerblockers of maxi-K channels than Compound C. 10 nM Compound D causedless than a 50% reduction in channel open probability, and completeblock was not observed at 1 μM. 10 nM of Compound A blocked a smallfraction of channel activity, 100 nM blocked approximately one half ofthe channel activity, and 1 μM blocked nearly all of the channelactivity. 1 μM Compound E had no significant effect on channel openprobability, while 10 μM caused an incomplete block of channel activityslowly increasing over 5-10 minutes. Compound F caused approximately a50% decrease in channel activity at 10 nM. Compound G at 0.1 nM blocked82% of channels, at 1 nM blocked 98.6%, and at 10 nm caused >99% block.The data is summarized in the table below:

COMPOUND [50% CHANNEL BLOCK] A 100 nm (approx.) B <1 nM C <10 nM D 1 μM(approx.) E >10 μM F 10 nM (approx.) G <0.1 nM (approx.)

EXAMPLE 5

Biochemical Experiments

Methods:

The interaction of [¹²⁵I]ChTX with bovine aortic sarcolemma membranevesicles was determined under conditions as described (Vazquez et al.,1989, J. Biol. Chem. 264, 20902-20909). Briefly, sarcolemma membranevesicles were incubated in 12×75 polystyrene tubes with ca. 25 pM[¹²⁵I]ChTX (2200 Ci/mmol), in the absence or presence of test compound,in a media consisting of 20 mM NaCl, 20 mM Tris-HCl pH 7.4, 0.1% bovineserum albumin, 0.1% digitonin. Nonspecific binding was determined in thepresence of 10 nM ChTX. Incubations were carried out at room temperatureuntil ligand binding equilibrium is achieved at ca. 90 min. At the endof the incubation period, samples were diluted with 4 mL ice-cold 100 mMNaCl, 20 mM Hepes-Tris pH 7.4 and filtered through GF/C glass fiberfilters that have been presoaked in 0.5% polyethylenimine. Filters wererinsed twice with 4 mL ice-cold quench solution. Radioactivityassociated with filters was determined in a gamma counter. Specificbinding data in the presence of each compound (difference between totalbinding and nonspecific binding) was assessed relative to an untreatedcontrol.

Results:

Compound D (hydroxypaspalinic acid) did not have any effect on bindingin the range of concentrations from 1 nM to 100 μM. Compound E(methylhydroxypaspalinate) did not affect [¹²⁵I]ChTX binding from 1 nMto 100 μM. Compound A and Compound C inhibited binding by 2% and 29%,respectively, when tested at 10 μM.

The effect of Compound F, paxizoline, was investigated by increasing theconcentration of compound in the assay from 1 nM to 100 μM. Thiscompound produced two opposite effects on toxin binding. In the range of50 nM to 2 μM, there was a small increase in the amount of toxin boundto its receptor. At concentrations above 5 μM, Compound F caused aconcentration-dependent inhibition of binding. It appears that themaximum level of inhibition saturates at ca. 32% control.

Compound G inhibited binding by 35% at 10 μM.

EXAMPLE 6

The activity of the compounds can also be quantified by the followingassays.

A. Maxi-K Channel

The identification of inhibitors of the Maxi-K channel can beaccomplished using Aurora Biosciences technology, and is based on theability of expressed Maxi-K channels to set cellular resting potentialafter transient transfection of both α and β subunits of the channel inTsA-201 cells. In the absence of inhibitors, cells display ahyperpolarized membrane potential, negative inside, close to E_(K) (−80mV) which is a consequence of the activity of the Maxi-K channel.Blockade of the Maxi-K channel will cause cell depolarization. Changesin membrane potential can be determined with voltage-sensitivefluorescence resonance energy transfer (FRET) dye pairs that use twocomponents, a donor coumarin (CC₂DMPE) and an acceptor oxanol(DiSBAC₂(3)). Oxanol is a lipophilic anion and distributes across themembrane according to membrane potential. Under normal conditions, whenthe inside of the cell is negative with respect to the outside, oxanolis accumulated at the outer leaflet of the membrane and excitation ofcoumarin will cause FRET to occur. Conditions that lead to membranedepolarization will cause the oxanol to redistribute to the inside ofthe cell, and, as a consequence, to a decrease in FRET. Thus, the ratiochange (donor/acceptor) increases after membrane depolarization.

Transient transfection of the Maxi-K channel in TsA-201 cells can becarried out as previously described (Hanner et al. (1998) J. Biol. Chem.273, 16289-16296) using FUGENE6™ as the transfection reagent. Twentyfour hours after transfection, cells are collected in Ca²⁺-Mg²⁺-freeDulbecco's phosphate-buffered saline (D-PBS), subjected tocentrifugation, plated onto 96-well poly-d-lysine coated plates at adensity of 60,000 cells/well, and incubated overnight. The cells arethen washed 1× with D-PBS, and loaded with 100 μl of 4 μM CC₂DMPE-0.02%pluronic-127 in D-PBS. Cells are incubated at room temperature for 30min in the dark. Afterwards, cells are washed 2× with D-PBS and loadedwith 100 μl of 6 μM DiSBAC₂(3) in (mM): 140 NaCl, 0.1 KCl, 2 CaCl₂, 1MgCl₂, 20 Hepes-NaOH, pH 7.4, 10 glucose. Test compounds are dilutedinto this solution, and added at the same time. Cells are incubated atroom temperature for 30 min in the dark.

Plates are loaded into a voltage/ion probe reader (VIPR) instrument, andthe fluorescence emission of both CC₂DMPE and DiSBAC₂(3) are recordedfor 10 sec. At this point, 100 μl of high-potassium solution (mM): 140KCl, 2 CaCl₂, 1 MgCl₂, 20 Hepes-KOH, pH 7.4, 10 glucose are added andthe fluorescence emission of both dyes recorded for an additional 10sec. The ratio CC₂DMPE/DiSBAC₂(3), before addition of high-potassiumsolution equals 1. In the absence of any inhibitor, the ratio afteraddition of high-potassium solution varies between 1.65-2.0. When theMaxi-K channel has been completely inhibited by either a known standardor test compound, this ratio remains at 1. It is possible, therefore, totitrate the activity of a Maxi-K channel inhibitor by monitoring theconcentration-dependent change in the fluorescence ratio.

B. Electrophysiological Assays of Compound Effects on High-conductanceCalcium-activated Potassium Channels

Human Non-pigmented Ciliary Epithelial Cells

The activity of high-conductance calcium-activated potassium (maxi-K)channels in human non-pigmented ciliary epithelial cells can bedetermined using electrophysiological methods. Currents through maxi-Kchannels can be recorded in the inside-out configuration of the patchclamp technique, where the pipette solution faces the extracellular sideof the channel and the bath solution faces the intracellular side.Excised patches contain one to about fifty maxi-K channels. Maxi-Kchannels can be identified by their large single channel conductance(250-300 pS), and by sensitivity of channel gating to membrane potentialand intracellular calcium concentration. Membrane currents are recordedusing standard electrophysiological techniques. Glass pipettes (Garner7052) are pulled in two stages with a Kopf puller (model 750), andelectrode resistance is 1-3 megohms when filled with saline. Membranecurrents are recorded with EPC9 (HEKA Instruments) or Axopatch iD (AxonInstruments) amplifiers, and digital conversion was done with ITC-16interfaces (Instrutech Corp). Pipettes are filled with (mM); 150 KCl, 10Hepes, 1 MgCl₂, 0.01 CaCl₂, 3.65 KOH, pH 7.20. The bath (intracellular)solution is identical, except, in some cases, calcium is removed, 1 mMEGTA is added and 20 mM KCl is replaced with 20 mM KF to eliminatecalcium to test for calcium sensitivity of channel gating. Drugs areapplied to the intracellular side of the channel by bath perfusion.

Human non-pigmented ciliary epithelial cells can be grown in tissueculture as described (Martin-Vasallo, P., Ghosh, S., and Coca-Prados,M., 1989, J. Cell. Physiol. 141, 243-252), and plated onto glass coverslips prior to use. High resistance seals (>1 Gohm) are formed betweenthe pipette and cell surface, and inside out patches are excised. Maxi-Kchannels in the patch are identified by their gating properties; channelopen probability increases in response to membrane depolarization andelevated intracellular calcium. In patches used for pharmacologicalanalysis, removing intracellular calcium eliminates voltage-gatedcurrents. Maxi-K currents are measured after depolarizing voltage stepsor ramps that cause channel opening.

1. A method for treating macular edema or macular degeneration whichcomprises administering to a patient in need of such treatment apharmaceutically effective amount of a compound of structural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein, R¹ is: (a) H,

R² is: (a) CO₂C₁₋₆alkyl, (b) H, (c) OH, or R² and R⁷ are taken togetherto form an oxirane ring when b is a single bond; R³ is: (a) H, or (b)(C═O)OC₁₋₆alkyl; R⁵ is: (a) H, (b) OH, or (c) OC₁₋₆alkyl; R⁷ is H, OC₁₋₆alkyl or R⁷ and R² are taken together to form an oxirane ring when b isa single bond; Y is: (a) H, (b) OH, (c) OC₁₋₆alkyl, or Y and R⁸ arejoined such that one of the following rings is formed:

R⁸ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl; and - - - is a double bondoptionally present at a or b or at both a and b.
 2. The method accordingto claim 1 wherein the compound of formula I is applied as a topicalformulation.
 3. The method according to claim 1 wherein the compound isselected from:

and pharmaceutically acceptable salts, enantiomers, diastereomers andmixtures thereof.